Roche Diagnostics Operations, Inc.

Hoffmann-la Roche Inc.

Roche Diagnostics Gmbh

F. Hoffmann-la Roche Ag

Christine Jung

Harald Sobek

Rainer Mueller

Alfred Engel

Michael Greif

Bernhard Suppmann

Marco Thomann

Sebastian Malik

Helmut Schwab

Dietmar Reusch

Bernd Nidetzky

Christiane Luley

Tibor Czabany

Doris Ribitsch

Katharina Schmoelzer

Sabine Zitzenbacher

Markus Haberger

The present disclosure is directed to the use of certain glycosyltransferase variants having N-terminal truncation deletions. Contrary to previous findings certain truncations were found to exhibit sialidase enzymatic activity, particularly a variant of human sialyltransferase (hST6Gal-I) with a truncation deletion involving the first 89 N-terminal amino acids of the respective wild-type polypeptide. A fundamental finding documented in the present disclosure is that there exists a variant of this enzyme which is capable of catalyzing transfer of a glycosyl moiety as well as hydrolysis thereof. Thus, disclosed is a specific exemplary variant of mammalian glycosyltransferase, nucleic acids encoding the same, methods and means for recombinantly producing the variant of mammalian glycosyltransferase and use thereof, particularly for sialylating in a quantitatively controlled manner terminal acceptor groups of glycan moieties being part of glycoproteins such as immunoglobulins.

Quantitative control of sialylation

The present disclosure is directed to the use of certain glycosyltransferase variants having N-terminal truncation deletions. It was found that the combination of two different truncation variants of human &#x3b2;-galactoside-&#x3b1;-2,6-sialyltransferase I (hST6Gal-I) exhibited different specific sialyltransferase enzymatic activities. In one example, under conditions wherein the first variant &#x394;89 hST6Gal-I catalyzed formation of bi-sialylated target molecules the second variant &#x394;108 hST6Gal-I catalyzed formation of mono-sialylated target molecules. Thus, disclosed are variants of mammalian glycosyltransferase, nucleic acids encoding the same, methods and means for recombinantly producing the variants of mammalian glycosyltransferase and use thereof, particularly for sialylating in a quantitatively controlled manner terminal acceptor groups of glycan moieties being part of glycoproteins such as immunoglobulins.

Quantitative control of sialylation and specific mono-sialylation

The current invention comprises a method for producing an immunoglobulin or immunoglobulin fragment with defined glycostructure comprising the following steps: a) providing an affinity chromatography column eluate containing the immunoglobulin or immunoglobulin fragment, b) incubating the affinity chromatography column eluate with (&#x3b1;1,3)galactosidase of plant origin, e.g. from green coffee beans (EC 3.2.1.22), c) applying the incubated affinity chromatography column eluate to a protein A chromatography material and recovering the immunoglobulin or immunoglobulin fragment from the protein A chromatography material and thereby producing an immunoglobulin or immunoglobulin fragment with defined glycostructure.

Enzymatic antibody processing

Growing community of inventors

Iffeldorf, Germany

Christine Jung